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Diltiazem
Molecular formula: C22H26N2O4S Molecular weight: 414.5 CAS Registry No.: 42399-41-7 (diltiazem), 33286-22-5 (diltiazem hydrochloride)

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 200 fxL 500 ng/mL 4-methylpropranolol in solvent + 1 mL buffer, mix briefly, add 5 mL MTBE, shake vigorously for 10 min, centrifuge at 2500 g for 10 min. Remove 4 mL of the organic layer and evaporate it to dryness using a vortex evaporator at 30, reconstitute the residue in 2 mL hexane and 200 |JLL solvent, vortex for 2 min, discard the upper hexane layer, wash again with 2 mL hexane, inject a 100 jxL aliquot of the aqueous phase. (Buffer was 200 mM K2HPO4 adjusted to pH 10 with 5 M KOH. Solvent was MeCN: MeOH: 10 mM pH 2 sulfuric acid 10:45:45 containing 56 mM sodium octanesulfonate. MTBE was stored over activated charcoal and filtered (Whatman No. 2v) immediately before use. Hexane was purified by stirring 4 volumes hexane with 1 volume concentrated sulfuric acid overnight then washing twice with 1 volume water.) HPLCVARIABLES Guard column: 20 X 4.6 5 fxm Suplex pKb-100 (Supelco) Column: 150 X 4.6 5 [im Suplex pKb-100 (Supelco) Mobile phase: MeCN: MeOH .10 mM pH 2 sulfuric acid 10:45:45 containing 10 mM sodium octanesulfonate Flow rate: 1 Injection volume: 200 Detector: UV 237 CHROMATOGRAM Retention time: 10.3 Internal standard: 4-methylpropranolol (Wyeth-Ayerst) (17.1) Limit of quantitation: 2 ng/mL OTHER SUBSTANCES Extracted: metabolites, quinidine (F ex 247 em 270) KEYWORDS plasma REFERENCE
Carignan, G.; Carrier, K.; Laganiere, S.; Lessard, M. Simultaneous determination of diltiazem and quinidine in human plasma by liquid chromatography. J.Chromatogr.B, 1995, 672, 261-269

SAMPLE Matrix: blood Sample preparation: Make serum alkaline with 10% sodium carbonate, extract with diisopropyl ether (Caution! Diisopropyl ether readily forms explosive peroxides!). Remove the organic layer and extract it with 10 mM HCl5 inject an aliquot of the aqueous layer.

HPLCVARIABLES Column: 150 X 4.6 5 |xm Supelcosil LC-CN Mobile phase: MeCN: water: 500 mM KH2PO4 36:62:2 Flow rate: 1.8 Detector: UV 210 CHROMATOGRAM Limit of detection: 10 ng/mL OTHER SUBSTANCES Extracted: mexiletine, propafenone KEYWORDS serum REFERENCE
Kunicki, RK.; Sitkiewicz, D. High-performance liquid chromatographic determination of some antiarrhythmic drugs using cyanopropyl derivatized silica phase (Abstract 43). Ther.Drug Monit., 1995,17, 394

SAMPLE Matrix: blood Sample preparation: 2 mL Whole blood or plasma + 2 inL buffer H 5 mL chloroform: isopropanol: n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.) HPLCVARIABLES Column: 300 X 3.9 4 |xm NovaPack C18 Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.) Column temperature: 30 Flow rate: 0.8 Injection volume: 50 Detector: UV 238 CHROMATOGRAM Retention time: 5.90 Limit of detection: <120 ng/mL OTHER SUBSTANCES Extracted: acebutolol, acenocoumarol, acepromazine, aceprometazine, acetaminophen, ajmaline, albuterol, alimemazine, alminoprofen, alpidem, alprazolam, alprenolol, amisulpride, amitriptyline, amodiaquine, amoxapine, aspirin, atenolol, benazepril, benperidol, benzocaine, benzoylecgonine, bepridil, betaxolol, bromazepam, brompheniramine, bumadizone, bupivacaine, buprenorphine, buspirone, caffeine, carbamazepine, carbinoxamine, carpipramine, carteolol, celiprolol, cetirizine, chlorambucil, chlordiazepoxide, chlormezanone, chlorophenacinone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, cibenzoline, cicletanine, clemastine, clobazam, clomipramine, clonazepam, clonidine, clorazepate, clozapine, cocaine, codeine, colchicine, cyamemazine, cyclizine, cycloguanil, cyproheptadine, cytarabine, dacarbazine, daunorubicin, debrisoquine, demexiptiline, desipramine, dextromethorphan, dextromoramide, dextropropoxyphene, diazoxide, diclofenac, dihydralazine, diphenhydramine, dipyridamole, disopyramide, dosulepine, doxepin,

doxylamine, droperidol, ephedrine, estazolam, etodolac, fenfluramine, fenoprofen, fentiazac, flecainide, floctafenine, flumazenil, flunitrazepam, fluoxetine, fluphenazine, flurbiprofen, fiuvoxamine, glibenclamide, glipizide, glutethimide, histapyrrodine, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, iproniazid, ketamine, ketoprofen, labetalol, levomepromazine, lidocaine, lidoflazine, lisinopril, loperamide, loprazolam, loratadine, lorazepam, loxapine, maprotiline, medazepam, mefenamie acid, mefenidramine, mefloquine, melphalan, meperidine, mephenesin, mephentermine, mepivacaine, metapramine, metformin, methadone, methaqualone, methocarbamol, methotrexate, metipranolol, metoclopramide, metoprolol, mexiletine, midazolam, minoxidil, moclobemide, moperone, morphine, nadolol, nalbuphine, nalorphine, naloxone, naltrexone, naproxen, nialamide, nifedipine, niflumic acid, nimodipine, nitrazepam, nizatidine, nomifensine, nortriptyline, omeprazole, opipramol, oxazepam, oxprenolol, penbutolol, penfluridol, pentazocine, phencyclidine, phenobarbital, phenol, phenylbutazone, pimozide, pindolol, pipamperone, piroxicam, prazepam, prazosin, prilocaine, procainamide, procarbazine, proguanil, promethazine, propafenone, propranolol, protriptyline, pyrimethamine, quinidine, quinine, quinupramine, ranitidine, ritodrine, secobarbital, sotalol, strychnine, sulfinpyrazole, sulindac, sulpride, sultopride, suriclone, temazepam, tenoxicam, terbutaline, terfenadine, tetracaine, tetrazepam, thiopental, thioproperazine, thioridazine, tianeptine, tiapride, tiaprofenic acid, ticlopidine, timolol, tioclomarol, tofisopam, tolbutamide, toloxatone, trazodone, triazolam, trifluoperazine, trifluperidol, trimipramine, triprolidine, tropatenine, verapamil, viloxazine, vincristine, vindesine, warfarin, yohimbine, zolpidem, zopiclone, zorubicine Interfering: aconitine, astemizole, bisoprolol, diazepam, diltiazem, glibornuride, haloperidol, medifoxamine, mianserine, nicardipine, nitrendipine, ramipril, reserpine, vinblastine KEYWORDS whole blood; plasma REFERENCE
Tracqui, A.; Kintz, R; Mangin, P. Systematic toxicological analysis using HPLC/DAD. J.Forensic ScL, 1995, 40, 254-262

SAMPLE Matrix: blood Sample preparation: Wash a Lida 100 mg C18 SPE column with 3 mL MeCN then 3 mL 100 mM ammonium dihydrogen phosphate. 1 mL Plasma + 20 JJLL 7.5 jjLg/mL propyldiltiazem in MeOH + 500 |xL 100 mM ammonium dihydrogen phosphate, vortex, add to SPE column, wash with 2 mL MeCN: water 20:80, wash with 1 mL MeCN: water 40:60, air dry column for 30 s, elute with 500 (xL MeCN: 100 mM ammonium dihydrogen phosphate 80:20 containing 0.06% triethylamine (final pH 6.8). Evaporate eluate to dryness under nitrogen at 40-45, dissolve residue in 250 |xL MeCN: 50 mM KH2PO4 pH 2.9 20: 80, inject 100 |xL aliquot. HPLC VARIABLES Guard column: 20 X 4.6 40 ^m Pelliguard LC8 Column: 150 X 4.6 5 \xm Hypersil C8 BDS Mobile phase: MeCN: 50 mM KH2PO4 adjusted to pH 2.9 with phosphoric acid triethylamine 398:600:2 Flow rate: 1 Injection volume: 100 Detector: UV 238 CHROMATOGRAM Retention time: 6 Internal standard: propyldiltiazem (8) Limit of quantitation: 2.5 ng/mL

OTHER SUBSTANCES Simultaneous: metabolites KEYWORDS plasma; SPE REFERENCE

Ascalone, V.; Locatelli, M.; Malavasi, B. Determination of diltiazem and its main metabolites in human plasma by automated solid-phase extraction and high-performance liquid chromatography: a new method overcoming instability of the compounds and interference problems. J.Chromatogr.B, 1994, 657, 133-140

SAMPLE Matrix: blood Sample preparation: 1 mL Plasma + 5 mL MTBE + 40 |xL 10 |xg/mL verapamil in MeOH, vortex, centrifuge at 3000 g for 15 min. Remove the supernatant and add it to 80 |xL 50 mM sulfuric acid, vortex, centrifuge, inject a 25 uX aliquot of the lower aqueous phase. HPLC VARIABLES Guard column: 42 X 3 30-35 |xm CO-PELL ODS Column: 250 X 4.6 10 |xm Econosil-CN Mobile phase: MeOH: 50 mM ammonium dihydrogen phosphate: triethylamine 45:55: 0.25, pH adjusted to 5.0 with 1 M phosphoric acid Injection volume: 25 Detector: UV 237 CHROMATOGRAM Internal standard: verapamil KEYWORDS plasma; pharmacokinetics REFERENCE
Bialer, M.; Hadad, S.; Golomb, G.; Barel, S.; Samara, E.; Abu Salach, O.; Berkman, N.; Danenberg, H.D.; Ben David, J.; Caron, D. Pharmacokinetic analysis of two new sustained-release products of diltiazem designed for twice- and once-daily treatment. Biopharm.Drug Dispos., 1994, 15, 45-52

SAMPLE Matrix: blood Sample preparation: 1 mL Serum + 200 |xL 1 M NaOH, vortex 30 s, dilute to 5 mL with MeCN, vortex 2 min, centrifuge at 2200 g for 5 min, inject a 100 |JLL aliquot of supernatant. HPLC VARIABLES Guard column: 23 X 3.6 10 |xm Waters C8 Column: 300 X 4.1 10 jjum LiChrosorb RP-8 Mobile phase: MeCN: 10 mM Na2HPO4 + 0.1% triethanolamine 40:60, pH adjusted to 3.0 0 . 1 with 85% phosphoric acid Flow rate: 1.2 Injection volume: 100 Detector: UV 237 CHROMATOGRAM Retention time: 7

Limit of detection: 2.5 ng/mL Limit of quantitation: 10 ng/mL KEYWORDS serum REFERENCE
Chaudhary, R.S.; Gangwal, S.S.; Avachat, M.K.; Shah, Y.N.; Jindal, K.C. Determination of diltiazem hydrochloride in human serum by high-performance liquid chromatography. J.Chromatogr., 1993, 614, 261-266

SAMPLE Matrix: blood Sample preparation: 0.5 mL Plasma + hexane: 2-propanol 98:2, stir 15 min, centrifuge 1500 g 10 min. Remove organic layer and evaporate it to dryness under a stream of nitrogen at 50. Reconstitute residue in 200 jutL mobile phase, vortex 1 min, inject 20 juiL aliquot. HPLCVARIABLES Guard column: 18 X 0.4 5 *xm Ultron ES-OVM Column: 150 X 4.6 5 |x m Ultron ES-OVM (ovomucoid chemically bonded to aminopropylsilica gel) Mobile phase: EtOH: 20 mM KH2PO4 3:97, pH adjusted to 4.5 with phosphoric acid or KOH Flow rate: 1 Injection volume: 20 Detector: UV 237 CHROMATOGRAM Retention time: 3.5 (cis-(+)-diltiazem), 7.5 (cis-(-)-diltiazem) Limit of detection: 64 ng/mL KEYWORDS plasma; chiral; effect of organic modifier and pH on retention time and separation is discussed REFERENCE
Rosell, G.; Camacho, A.; Parra, P. Direct enantiomeric separation of cis-()diltiazem in plasma by highperformance liquid chromatography with ovomucoid column. J.Chromatogr., 1993, 619, 87-92

SAMPLE Matrix: blood Sample preparation: 2 mL Plasma + 100 JULL 3 |xg/mL propranolol hydrochloride in water + 6 mL MTBE, shake 15 min, centrifuge at 1500 g for 15 min. Remove organic layer and add it to 100 |xL 0.05 M sulfuric acid, shake 15 min, centrifuge at 1500 g at 4 for 10 min, discard organic layer, inject 50 |xL aliquots of aqueous layer. HPLCVARIABLES Column: 100 X 2 5 y.m ODS Hypersil Mobile phase: MeCN: 10 mM Na2HPO4 40:60 containing 40 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide, adjusted to pH 2 with orthophosphoric acid Flow rate: 0.5 Injection volume: 50 Detector: UV 240

CHROMATOGRAM

Retention time: 6 Internal standard: propranolol (10.5) Limit of detection: 1 ng/mL OTHER SUBSTANCES Simultaneous: metabolites
KEYWORDS

plasma; microbore
REFERENCE
Zoest, A.R.; Hung, C.T.; Wanwimolruk, S. Diltiazem: a sensitive HPLC assay and application to pharmacokinetic study. J.Liq.Chromatogr., 1992, 15, 1277-1287

SAMPLE

Matrix: blood Sample preparation: 2 mL Plasma + 100 |xL 6 jxg/mL imipramine in 10 mM HCl + 200 |JLL 10% ammonium carbonate (final pH 8.7), vortex gently, add 5 mL MTBE, extract (Vibrax VXR2) for 20 min, centrifuge at 4 at 1720 g for 10 min, remove the organic layer. Add 5 mL dichloromethane to the aqueous layer, shake for 20 min on a reciprocating shaker, centrifuge at 0 at 1720 g for 10 min. Combine the organic layers and evaporate them to dryness under a stream of nitrogen at 50, reconstitute the residue in 100 JJLL 10 mM HCl, wash with 2 mL MTBE, wash with 2 mL hexane, inject a 3-20 |xL aliquot of the aqueous layer.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere-ODS C18 Mobile phase: MeCN: MeOH: 40 mM ammonium acetate 24:40:36 containing 0.04% triethylamine, pH adjusted to 7.3 with glacial acetic acid Flow rate: 1.2 Injection volume: 3-20 Detector: UV 237
CHROMATOGRAM

Retention time: 13.0 Internal standard: imipramine (17.9)

OTHER SUBSTANCES

Extracted: metabolites Simultaneous: alprazolam, amitriptyline, desipramine, loxapine, nortriptyline Noninterfering: clomipramine
KEYWORDS

plasma; pharmacokinetics
REFERENCE
Yeung, P.K.F.; Montague, T.J.; Tsui, B.; McGregor, C. High-performance liquid chromatographic assay of diltiazem and six of its metabolites in plasma: application to a pharmacokinetic study in healthy volunteers. J.Pharm.ScL, 1989, 78, 592-597

SAMPLE

Matrix: blood Sample preparation: 500 u-L Serum + 250 (xL di-iso-propyl ether: n-butyl alcohol 7:3 containing 800 ng/mL minaprine, centrifuge 2 min, shake, centrifuge 5 min, inject 50 u,L aliquot of top organic layer.

HPLCVARIABLES Guard column: 30 X 4.6 5 |xm Brownlee cyano spheri-5 Column: 250 X 4.6 5 |xm Altex ultrasphere cyano Mobile phase: MeCN: THF: water: 2 M ammonium formate (pH 4.0) 700:100:195:5 Column temperature: 20 Flow rate: 1.5 Injection volume: 50 Detector: UV 240 CHROMATOGRAM Retention time: 6 Internal standard: minaprine (5.5) Limit of detection: 20 ng/mL OTHER SUBSTANCES Simultaneous: amitriptyline, nortriptyline Also analyzed: amiodarone, clomipramine, desipramine, haloperidol, imipramine, propafenone, verapamil KEYWORDS serum REFERENCE
Mazzi, G. Simple and practical high-performance liquid chromatographic assay of some tricyclic drugs, haloperidol, diltiazem, verapamil, propafenone, and amiodarone. Chromatographia, 1987, 24, 313316

SAMPLE Matrix: blood, tissue Sample preparation: Homogenize liver at 20 mg/mL in 50 mM pH 7.4 Tris-HCl buffer. 200 |JLL Plasma or 250 u,L liver homogenate + 250 ng IS + pH 7.3 ammonium phosphate buffer + MTBE, extract. Remove the organic layer and add it to 250 J U L L 50 mM phosphoric acid, extract. Remove the aqueous layer and add it to 100 |xL MeCN, inject an aliquot. HPLCVARIABLES Column: 100 X 4.6 3 jxm Chromegabond (ES Industries) Mobile phase: MeCN: 100 mM sodium perchlorate containing 50 mM phosphoric acid 34: 66 Column temperature: 30 Flow rate: 1 Detector: UV 237 CHROMATOGRAM Retention time: 4.5 Internal standard: N-methyl-N-ethyldiltiazem (9.5) Limit of quantitation: 10 ng/mL OTHER SUBSTANCES Extracted: metabolites KEYWORDS rat; plasma; liver; pharmacokinetics REFERENCE
Los, L.E.; Welsh, D.A.; Herold, E.G.; Bagdon, W.J.; Zacchei, A.G. Gender differences in toxicokinetics, liver metabolism, and plasma esterase activity: Observations from a chronic (27-week) toxicity study of enalapril/diltiazem combinations in rats. Drug Metab.Dispos., 1996, 24, 28-33

SAMPLE

Matrix: bulk Sample preparation: Inject a 10 J U L L aliquot of a 1 mg/mL diltiazem solution containing 1 mg/mL IS.
HPLCVARIABLES

Column: 250 X 4.6 10 \xm Chiralcel OF cellulose tris(4-chlorophenylcarbamate) Mobile phase: Hexane: isopropanol 50:50 containing 0.1% diethylamine Column temperature: 30 Flow rate: 1 Injection volume: 20 Detector: UV 254
CHROMATOGRAM

Retention time: 9 ((-)-trans), 11 ((-)-cis), 15 ((+)-trans), 19 ((+)-cis) Internal standard: (+)-cis-5-acetyl-2,3-dihydro-3-hydroxy-2-(p-methoxyphenyl)-l,5-benzothiazepin-4(5H)-one (acetylthiazepin) (24)
KEYWORDS

chiral
REFERENCE
Ishii, K.; Minato, K.; Nakai, H.; Sato, T. Simultaneous assay of four stereoisomers of diltiazem hydrochloride. Application to in vitro chiral inversion studies. Chromatographia, 1995, 41, 450-454

SAMPLE

Matrix: formulations Sample preparation: Powder tablets, weigh out powder equivalent to about 50 mg diltiazem, dissolve in 100 mL MeOH, filter. Remove 5 mL aliquot, add 5 mL 0.5 mg/mL cyproheptadine hydrochloride in MeOH, make up to 50 mL with water, inject 50 |xL aliquot.
HPLCVARIABLES

Column: 250 X 4.6 Rexchrome ODS Mobile phase: MeCN: MeOH: 50 mM KH2PO4 25:20:55 Flow rate: 2 Injection volume: 50 Detector: UV 240
CHROMATOGRAM

Retention time: 6 Internal standard: cyproheptadine (8)

KEYWORDS

tablets; stability-indicating
REFERENCE
Shivram, K.; Shah, A.C.; Newalkar, B.L.; Kamath, B.V. Stability indicating high-performance liquid chromatographic method for the assay of diltiazem hydrochloride in tablets. J.Liq.Chromatogr., 1992, 15, 2417-2422

SAMPLE Matrix: perfusate

HPLCVARIABLES Column: 100 X 8 4 |xm Novapak C18 Mobile phase: MeCN: 0.092% phosphoric acid containing 0.2% triethylamine 26:74 Flow rate: 2 Detector: UV 214 CHROMATOGRAM Internal standard: lidocaine Limit of quantitation: 10 ng/mL OTHER SUBSTANCES Simultaneous: metabolites, diphenhydramine Also analyzed: bupivacaine KEYWORDS rat; liver REFERENCE
Hussain, M.D.; Tarn, Y.K.; Gray, M.R.; Coutts, K.T. Kinetic interactions of lidocaine, diphenhydramine, and verapamil with diltiazem: A study using isolated perfused rat liver. Drug Metab.Dispos., 1994, 22, 530-536

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 5 ixm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B) Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric acid, make up to 1 L.) Flow rate: 0.6 Injection volume: 25 Detector: UV 229 CHROMATOGRAM Retention time: 11.10 (A), 5.42 (B) OTHER SUBSTANCES Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ievorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-

zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxyeodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone KEY WORDS some details of plasma extraction REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology. J.Chromatogr.A, 1995, 692, 103-119

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 Zorbax RX Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:100 for 5 min. Column temperature: 30 Flow rate: 2 Detector: UV 210 OTHER SUBSTANCES Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid,

medazepam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, triproilidine, tropacocaine, tyramine, verapamil, vincamine, warfarin, yohimbine, zoxazolamine REFERENCE
Hill, D. W.; Kind, A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs. J.Anal.ToxicoL, 1994, 18, 233-242

SAMPLE Matrix: solutions Sample preparation: Prepare a 50 |xg/mL solution in MeCN: water 40:60, inject an aliquot. HPLCVARIABLES Column: 250 X 4.6 3 |xm silica (Phenomenex) Mobile phase: MeCN: 6.25 mM pH 3.0 phosphate buffer 40:60 Flow rate: 1 Injection volume: 50 Detector: UV 254 CHROMATOGRAM Retention time: 9.08 OTHER SUBSTANCES Also analyzed: atenolol, clonidine, metoprolol, nifedipine, prazosin, propranolol, verapamil REFERENCE
Simmons, B.R.; Stewart, J.T. HPLC separation of selected cardiovascular agents on underivatized silica using an aqueous organic mobile phase. J.Liq.Chromatogr., 1994, 17, 2675-2690

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 cellulose tris(3,5-dimethylphenylcarbamate)

Mobile phase: Hexane: isopropanol 90:10 Flow rate: 0.5 Detector: UV CHROMATOGRAM Retention time: 21 (-), 28 (+) KEYWORDS chiral REFERENCE
Okamoto, Y.; Aburatani, R.; Hatano, K.; Hatada, K. Optical resolution of racemic drugs by chiral HPLC on cellulose and amylose tris(phenylcarbamate) derivatives. J.Liq.Chromatogr., 1988, 11, 2147-2163

ANNOTATED BIBLIOGRAPHY

Higashidate, S.; Imai, K.; Prados, P.; Adachi-Akahane, S.; Nagao, T. Relations between blood pressure and plasma norepinephrine concentrations after administration of diltiazem to rats: HPLC-peroxyoxalate chemiluminescence determination on an individual basis. Biomed.Chromatogr., 1994, 8, 19-21 [plasma; extracted diltiazem, dopamine, epinephrine, norepinephrine; rat; chemiluminescence detection;SPE] Hussain, M.D.; Tarn, Y.K.; Gray, M.R.; Coutts, R.T. Mechanisms of time-dependent kinetics of diltiazem in the isolated perfused rat liver. Drug Metab.Dispos., 1994, 22, 36-42 [perfusate; extracted metabolites; LOQ 30 nM; bupivacaine (IS)] Ishii, K.; Minato, K.; Nishimura, N.; Miyamoto, T.; Sato, T. Direct chromatographic resolution of four optical isomers of diltiazem hydrochloride on a Chiralcel OF column. J.Chromatogr., 1994, 686, 9 3 100 [chiral; column temp 10-40] Rutledge, D.R.; Abadi, A.H.; Lopez, L.M. Liquid chromatographic determination of celiprolol, diltiazem, desmethyldiltiazem and deacetyldiltiazem in plasma using a short alkyl chain silanol deactivated column. J.Pharm.Biomed.AnaL, 1994, 12, 135-140 [extracted metabolites, celiprolol; LOD 3 ng/mL] Sigusch, H.; Henschel, L.; Kraul, H.; Merkel, U.; Hoffmann, A. Lack of effect of grapefruit juice on diltiazem bioavailability in normal subjects. Pharmazie, 1994, 49, 675-679 [pharmacokinetics; SPE; extracted metabolites; flurazepam (IS)] Rutledge, D.R.; Abadi, A.H.; Lopez, L.M.; Beaudreau, CA. High-performance liquid chromatographic determination of diltiazem and two of its metabolites in plasma using a short alkyl chain silanol deactivated column. J.Chromatogr., 1993, 615, 111-116 [plasma; extracted metabolites; imipramine IS; LOD 4 ng/mL; pharmacokinetics; interfering theophylline; simultaneous desipramine, propranolol, verapamil; non-interfering aspirin, atenolol, caffeine, ibuprofen, lidocaine, metoprolol, nifedipine] Hubert, P.; Chiap, P.; Crommen, J. Automatic determination of diltiazem and deacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC-Part I: optimization of the HPLC system and method validation. J.Pharm.Biomed.AnaL, 1991, 9, 877-882 Hubert, P.; Chiap, P.; Crommen, J. Automatic determination of diltiazem and deacetyldiltiazem in human plasma using liquid-solid extraction on disposable cartridges coupled to HPLC-Part II: optimization of liquid- solid extraction. J.Pharm.Biomed.AnaL, 1991, 9, 883-887 Ishii, K.; Banno, K.; Miyamoto, T.; Kakimoto, T. Determination of diltiazem hydrochloride enantiomers in dog plasma using chiral stationary-phase liquid chromatography. J.Chromatogr, 1991, 564, 338345 [dog; plasma; chiral; achiral; extracted metabolites; column temp 40] Jensen, B.H.; Larsen, C. Quantitation of diltiazem in human plasma by HPLC using an end-capped reversed-phase column. Acta Pharm.Nord., 1991, 3, 179-180 Leneveu, A.; Stheneur, A.; Bousquet, A.; Roux, A. Automated high-performance liquid chromatographic technique for determining diltiazem and its three main metabolites in serum. J.Liq.Chromatogr, 1991, 14, 3519-3530 [serum; extracted metabolites; SPE; pharmacokinetics; LOD 2.5 ng/mL] Bonnefous, J.L.; Boulieu, R. Comparison of solid-phase extraction and liquid-liquid extraction methods for liquid chromatographic determination of diltiazem and its metabolites in plasma.

J.Liq.Chromatogr, 1990, 13, 3799-3807 [extracted metabolites; plasma; SPE; propionyldeacetyldiltiazem (IS); LOD 5 ng/mL] Boulieu, R.; Bonnefous, J.L.; Ferry, S. Determination of diltiazem and its metabolites in plasma by high performance liquid chromatography J.Liq.Chromatogr., 1990, 13, 291-301 [plasma; extracted metabolites; propionyldeacetyldiltiazem (IS); LOD 5 ng/mL] Boulieu, R.; Bonnefous, J.L.; Ferry, S. Solid-phase extraction of diltiazem and its metabolites from plasma prior to high-performance liquid chromatography. J.Chromatogr., 1990, 528, 542-546 [plasma; propionyldeacetyldiltiazem (IS); SPE; extracted metabolites; LOD 0.3 ng; non-interfering diazepam, flunitrazepam, midazolam, nifedipine, pancuronium bromide, procainamide, propranolol, quinidine, verapamil] Johnson, K.E.; Pieper, J.A. An HPLC method for the determination of diltiazem and three of its metabolites in serum. J.Liq.Chromatogr., 1990, 13, 951-960 [extracted metabolites; serum; doxepin (IS); LOD 3 ng/mL; non-interfering carbamazepine, chlorpromazine, gallopamil, imipramine, lidocaine, prochlorperazine, quinidine, thioridazine, trimeprazine; pharmacokinetics] Parissi-Poulou, M.; Ismailos, G.; Macheras, P. Modified HPLC analysis of diltiazem in plasma for pharmacokinetic studies. Int.J.Pharm., 1990, 62, R13-R16 Shah, Y; Khanna, S.; Dighe, V.S.; Jindal, K.C. High-performance liquid chromatographic determination of diltiazem hydrochloride in tablets. Indian Drugs, 1990, 27, 363-364 Ververs, FF.T.; Schaefer, H.G.; Lefevre, J.F.; Lopez, L.M.; Derendorf, H. Simultaneous assay of propranolol, diltiazem and metabolites of diltiazem in human plasma by liquid chromatography. J.Pharm.Biomed.Anal, 1990, 8, 535-539 Yamahara, H.; Suzuki, T.; Mizobe, M.; Noda, K.; Samejima, M. In situ perfusion system for oral mucosal absorption in dogs. J.Pharm.ScL, 1990, 79, 963-967 [perfusate] Ascalone, V.; Flaminio, L. Automated high-performance liquid chromatography with column switching for on-line clean-up and analysis of diltiazem and metabolites in human plasma. J.Chromatogr., 1989, 495, 358-360 [plasma; column-switching; extracted metabolites; LOD 2 ng/mL; improved version of method in J.Chromatogr. 1987, 423, 239] Boucher, S.; Varin, F; Theoret, Y; Du Souich, P.; Caille, G. High-performance liquid chromatographic method for the determination of diltiazem and two of its metabolites in human plasma: application to a new sustained release formulation. J.Pharm.Biomed.Anal., 1989, 7, 1925-1930 Caille, G.; Dube, L.M.; Theoret, Y; Varin, F; Mousseau, N.; McGilveray, LJ. Stability study of diltiazem and two of its metabolites using a high performance liquid chromatographic method. Biopharm.Drug Dispos., 1989, 10, 107-114 Lacroix, P.M.; Beaulieu, N.; Cyr, T.D.; Lovering, E.G. High-performance liquid chromatography method for assay of diltiazem hydrochloride and its related compounds in bulk drug and finished tablets. J.Pharm.ScL, 1989, 78, 243-246 Rustum, A.M. Determination of diltiazem in human whole blood and plasma by high-performance liquid chromatography using a polymeric reversed-phase column and utilizing a salting-out extraction procedure. J.Chromatogr., 1989, 490:, 365-375 Zhao, H.; Chow, M.S.S. Analysis of diltiazem and desacetyldiltiazem in plasma using modified highperformance liquid chromatography: improved sensitivity and reproducibility. Pharm.Res., 1989, 6, 428-430 Dube, L.M.; Mousseau, N.; McGilveray, LJ. High-performance liquid chromatographic determination of diltiazem and four of its metabolites in plasma: evaluation of their stability. J.Chromatogr, 1988, 430, 103-111 Ascalone, V.; Dal Bo', L. Automated high-performance liquid chromatographic and column-switching technique for on-line clean-up and analysis of diltiazem in human plasma. J.Chromatogr., 1987, 423, 239-249 Bhamra, R.K.; Ward, A.E.; Holt, D.W. HPLC measurement of diltiazem and desacetyldiltiazem in serum or plasma. Biomed.Chromatogr, 1987, 2, 180-182 Hoglund, P.; Nilsson, L.G. Liquid chromatographic determination of diltiazem and its metabolites using trans isomers as internal standards, with dynamic modification of the solid phase by addition of an amine to the mobile phase. J.Chromatogr, 1987, 414, 109-120 Johnson, SM.; Wahba Khalil, S.K. An HPLC method for the determination of diltiazem and desacetyldiltiazem in human plasma. J.Liq.Chromatogr, 1987, 10, 673-685 [plasma; extracted desacetyld-

iltiazem; diazepam (IS); LOD 2 ng/mL; non-interfering atenolol, chlorthalidone, furosemide, hydralazine, methyldopa, pentoxifylline; also captopril, chlorothiazide, dipyridamole, disopyramide, isosorbide dinitrate, labetalol, metoprolol, pindolol, procainamide, propranolol, quinidine, warfarin] Montamat, S.C; Abernethy, D.R.; Mitchell, J.R. High-performance liquid chromatographic determination of diltiazem and its major metabolites, N-monodemethyldiltiazem and desacetyldiltiazem, in plasma. J.Chromatogr., 1987, 415, 203-207 Kinney, CD.; Kelly, J.G. Estimation of concentrations of diltiazem in plasma using normal-phase column liquid chromatography with ultraviolet detection. J.Chromatogr., 1986, 382, 377-381 Abernethy, D.R.; Schwartz, J.B.; Todd, E.L. Diltiazem and desacetyldiltiazem analysis in human plasma using high-performance liquid chromatography: improved sensitivity without derivation. J.Chromatogr., 1985, 342, 216-220 Goebel, K. J.; Kolle, E.U. High-performance liquid chromatographic determination of diltiazem and four of its metabolites in plasma. Application to pharmacokinetics. J.Chromatogr., 1985, 345, 355-363 Clozel, J.R; Caille, G.; Taeymans, Y.; Theroux, P.; Biron, P.; Trudel, F. High-performance liquid chromatographic determination of diltiazem and six of its metabolites in human urine. J.Pharm.Sci., 1984, 73, 771-773 Wiens, R.E.; Runser, D.J.; Lacz, J.R; Dimmitt, D.C. Quantitation of diltiazem and desacetyldiltiazem in dog plasma by high-performance liquid chromatography. J.Pharm.Sci., 1984, 73, 688-689 Verghese, C; Smith, M.S.; Aanonsen, L.; Pritchett, E.L.; Shand, D.G. High-performance liquid chromatographic analysis of diltiazem and its metabolite in plasma. J.Chromatogr., 1983, 272, 149-155 Hussain, M.D.; Tarn, Y.K.; Finegan, B.A.; Coutts, R.T. Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma. J.Chromatogr., 1992, 582, 203209 [plasma; benzylamphetamine (IS); extracted metabolites; pharmacokinetics; LOQ 5 ng/mL; non-interfering bupivacaine, diphenhydramine, lidocaine, metoprolol]

Doxazosin
Molecular formula: C23H25N5O5 Molecular weight: 451.5 CAS Registry No.: 74191 -85-8 (doxazosin), 77883-43-3 (doxazosin mesylate)

SAMPLE

Matrix: blood Sample preparation: Condition a Bond Elut C18 SPE cartridge with 1 mL MeOH and 1 mL water. 1 mL Plasma + 50 |JLL 1 |xg/mL propranolol in MeOH + 250 |JLL MeOH, agitate briefly, let stand for 10 min, centrifuge at 3000 g for 10 min. Add the supernatant to the SPE cartridge, wash with 1 mL MeOH: water 30:70, wash with 1 mL water, elute with 1 mL MeOH.acetic acid 99.5:0.5. Evaporate the eluate to dryness under a stream of air, reconstitute the residue in 150 |xL mobile phase, inject an aliquot.
HPLCVARIABLES

Column: 150 X 4.6 5 |xm Zorbax CN Mobile phase: MeOH: buffer 50:50 (Buffer was 10 mM perchloric acid and 1.8 mM sodium heptanesulfonate.) Flow rate: 1 Detector: F ex 245 em bandpass 320-390 (Corning 7-60 filter)
CHROMATOGRAM

Retention time: 5 Internal standard: propranolol (3.5) Limit of detection: 1 ng/mL

KEYWORDS

plasma; pharmacokinetics; SPE

REFERENCE
Jackman, G.P.; Colagrande, F.; Louis, W.J. Validation of a solid-phase extraction high-performance liquid chromatographic assay for doxazosin. J.Chromatogr., 1991, 566, 234-238

SAMPLE

Matrix: blood Sample preparation: 250 |xL Serum + 250 jxL 20 ng/mL prazosin in MeOH: water 30:70, vortex for 30 s, add 1.5 mL ethyl acetate, vortex for 15 s, centrifuge at 3000 rpm for 2 min. Remove the organic layer and evaporate it to dryness in a vortex evaporator at 40 for 10 min, reconstitute the residue in 200 jxL MeOH-.water 50:50, inject a 50 \xL aliquot.
HPLCVARIABLES

Guard column: 50 X 3.9 40 jim glass beads Column: 150 X 4.6 alumina-based reversed-phase gamma RP-I (ES Industries) Mobile phase: MeCN: MeOH: 25 mM pH 10.9 sodium carbonate 15:15:70 (At the end of each day flush with about 75 mL MeOH .water 50:50 until pH of effluent is neutral.) Flow rate: 2 Injection volume: 50 Detector: F ex 246 em 389 (cutoff filter)
CHROMATOGRAM Retention time: 4.9

Internal standard: prazosin (1.8) Limit of quantitation: 0.5 ng/mL KEYWORDS serum REFERENCE
Fouda, H.G.; Twomey, T.M.; Schneider, R.R Liquid chromatographic analysis of doxazosin in human serum with manual and robotic sample preparation. J.Chromatogr.Sci., 1988, 26, 570-573

SAMPLE Matrix: blood Sample preparation: 1 mL Whole blood + 5 mL diethyl ether, shake for 10 min, centrifuge at 2000 rpm for 5 min, freeze in acetone/dry ice. Remove the organic layer and add it to 100 |JLL 50 mM sulfuric acid, shake for 10 min, centrifuge at 2000 rpm for 5 min, inject a 20 |xL aliquot of the aqueous layer. HPLCVARIABLES Column: 250 X 4 5 |jim Spherisorb ODS Mobile phase: MeOH: water 55:45 containing 10 mM pentane sodium sulfate and 9 mM tetramethylammonium chloride, adjusted to pH 3.4 with glacial acetic acid Flow rate: 1.8 Injection volume: 20 Detector: F ex 254 em 400 (cut-off filter) CHROMATOGRAM Retention time: 9 Internal standard: doxazosin OTHER SUBSTANCES Extracted: trimazosin KEYWORDS whole blood; doxazosin is IS REFERENCE
Hughes, M.A.; Meredith, RA.; Elliott, H.L. The determination of trimazosin and its metabolite CP23445 in whole blood by high performance liquid chromatography using fluorescence detection. J.Pharmacol.Methods, 1984, 12, 29-34

SAMPLE Matrix: solutions HPLCVARIABLES Column: 150 X 4.6 12 |xm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine chemically bonded to silica (Regis) Mobile phase: MeCN: 100 mM pH 7.0 phosphate buffer 20:80 Flow rate: 1 Detector: UV 254 CHROMATOGRAM Retention time: k' 96.16

OTHER SUBSTANCES Also analyzed: acebutolol, alprenolol, antazoline, atenolol, betaxolol, bisoprolol, bopindolol, bupranolol, carteolol, celiprolol, chloropyramine, chlorpheniramine, cicloprolol, cimetidine, cinnarizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, esmolol, famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol, nizatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol, prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol, tiamenidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-14,304 REFERENCE
Kaliszan, R.; Nasal, A.; Turowski, M. Binding site for basic drugs on cti-acid glycoprotein as revealed by chemometric analysis of biochromatographic data. Biomed.Chromatogr., 1995, 9, 211215

SAMPLE Matrix: solutions HPLCVARIABLES Column: 125 X 5 5 |xm Spherisorb C8 Mobile phase: MeCN: water 25:45 containing 5 mM dibutylamine

Flow rate: 2
Detector: F ex 346 em 340 (filter) REFERENCE
Ferry, D.G.; Caplan, N.B.; Cubeddu, L.X. Interaction between antidepressants and alpha 1-adrenergic receptor antagonists on the binding to alpha 1-acid glycoprotein. J.Pharm.Sci., 1986, 75, 146-149

Doxycycline
Molecular formula: C22H24N2O8 Molecular weight: 444.4 CAS Registry No.: 564-25-0,17086-28-1 (monohydrate), 24390-14-5 (HCI monohydrate), 83038-87-3 (fosfatex), 24390-14-5 (hyclate)

SAMPLE Matrix: blood Sample preparation: 100 ^xL Serum + 50 JJLL 6% aqueous ascorbic acid + 50 ng demeclocycline in MeOH + 400 |xL buffer, vortex 30 s, add 3 mL ethyl acetate, vortex 5 min, centrifuge at 3000 rpm for 6 min. Remove organic layer and add it to 100 fxL 0.2% ascorbic acid in MeOH. Evaporate to dryness at 20 in a vortex evaporator, dissolve residue in 100 |xL mobile phase, inject entire amount. (Buffer was 2 M NaH2PO4 and 2 M Na2SO3, pH 6.1.) HPLCVARIABLES Guard column: 4 \xm Nova-Pak C18 Guard-Pak Column: 150 X 4.6 5 |xm Ultrabase C18 Mobile phase: MeCN: water adjusted to pH 2.5 with phosphoric acid 28:72 Flow rate: 1 Injection volume: 100 Detector: UV 350 CHROMATOGRAM Retention time: 4.2 Internal standard: demeclocycline (2.7) Limit of quantitation: 20 ng/mL KEYWORDS serum REFERENCE
Gastearena, L; Dios-Vieitez, M.C.; Segura, E.; Gofii, M.M.; Renedo, M.J.; Fos, D. Determination of doxycycline in small serum samples by liquid chromatography Application to pharmacokinetical studies on small laboratory animals. Chromatographia, 1993, 35, 524-526

SAMPLE Matrix: blood Sample preparation: 100 |JLL Plasma + 20 |JLL trifluoroacetic acid, mix 30 s in a whirl mixer, centrifuge at 5400 g for 5 min, inject supernatant (80 (xL). HPLCVARIABLES Guard column: 10 |xm Waters RP phenyl Column: 125 X 4.6 10 ^m Waters RP phenyl Mobile phase: MeCN: 10 mM phosphoric acid 30:70 Flow rate: 2 Injection volume: 80 Detector: UV 270 CHROMATOGRAM Retention time: 2.2 Limit of detection: 15 ng/mL

KEYWORDS plasma REFERENCE

Kramer-Horaczynska, F. High-performance liquid chromatographic procedures for the quantitative analysis of 15 tetracycline derivatives in small blood samples. J.Chromatogr.Sci., 1991, 29, 107-113

SAMPLE Matrix: blood, urine Sample preparation: Serum. 0.5 mL Serum + 0.5 mL MeCN:85% phosphoric acid: water 20:2:78, vortex, filter (10 000 or 30 000 Da cutoff) by centrifuging at 2200 g for 30 min, inject 10 |xL aliquot of filtrate. Urine. Dilute urine 5 to 10 times with MeCN: 85% phosphoric acid:water 20:1.7:78.3, vortex, filter (10 000 or 30 000 Da cutoff) by centrifuging at 2200 g for 30 min, inject 10 |xL aliquot of filtrate. HPLCVARIABLES Column: 220 X 4.6 phenyl Mobile phase: MeOH:MeCN:triethylamine:phosphoric acid:80 mM pH 2.4 sodium phosphate buffer 10:1.5:0.5:1.7:86.3 Column temperature: 50 Flow rate: 0.6-0.8 Injection volume: 10 Detector: UV 268; UV 345 CHROMATOGRAM Retention time: 8 Limit of detection: <10 ng/mL KEYWORDS serum; cow REFERENCE
Riond, J.L.; Hedeen, K.M.; Tyczkowska, K.; Riviere, J.E. Determination of doxycycline in bovine tissues and body fluids by high-performance liquid chromatography using photodiode array ultraviolet-visible detection. J.Pharm.ScL, 1989, 78, 44-47

SAMPLE Matrix: blood, urine Sample preparation: Serum. Condition a Bond-Elut C18 SPE cartridge with 1 volume MeOH and 2 volumes water. 1 mL Serum + 5 mL buffer, add to SPE cartridge, wash with 10 mL water, elute with 10 mL 10 mM phosphoric acid in MeCN. Evaporate eluate to dryness at 50 under a stream of nitrogen and resuspend residue in 1 mL water. Centrifuge at 10000 g for 1 min, inject 100 |xL aliquot. Urine. Activate a Bond-Elut C18 cartridge with 1 volume MeOH and 2 volumes water. 1 mL Urine + 5 mL buffer, add to cartridge, wash with 10 mL MeCN, elute with 10 mL 10 mM phosphoric acid in MeCN. Evaporate eluate to dryness at 50 under a stream of nitrogen and resuspend residue in 1 mL water. Centrifuge at 10 000 g for 1 min, inject 100 |xL aliquot. (Buffer was 0.1 M citric acid.0.2 M Na2HPO4 61.4:38.6 (Mcllvaines buffer) containing 0.1 M disodium EDTA.) HPLCVARIABLES Guard column: LiChrosorb RP-18 Column: 150 X 3.9 4 |xm Nova-Pak C18 Mobile phase: MeCN.acetic acid: 100 mM KH2PO4 75:150:125 (serum) or 65:150:125 (urine) Flow rate: 1

Injection volume: 100 Detector: UV 340 CHROMATOGRAM Retention time: 4 Limit of detection: 25 ng/mL KEYWORDS serum; SPE; protect from light with amber glassware REFERENCE
Sheridan, M.E.; Clarke, G.S. Improved high-performance liquid chromatographic determination of doxycycline in serum and urine using solid-phase extraction columns. J.Chromatogr., 1988, 434, 253258

SAMPLE Matrix: blood, urine Sample preparation: Serum. 500 |JLL Serum + 50 |xL 6% ascorbic acid in water + 50 |xL demeclocycline in MeOH/100 mM HCl + I m L buffer, mix for 30 s, add 6 mL ethyl acetate, rotate for 10 min, centrifuge at 3000 rpm for 6 min. Remove the organic layer and add it to 100 |xL 0.2% ascorbic acid in MeOH, evaporate to dryness under vacuum while vortexing, reconstitute the residue in 200 |xL mobile phase, mix, filter, keep in ice, inject a 20 |JLL aliquot. Urine. 100 |xL Urine + 50 |JLL 6% ascorbic acid in water + 50 |JLL demeclocycline in MeOH/100 mM HCl + 400 |JLL buffer, mix for 30 s, add 3 mL ethyl acetate, rotate for 10 min, centrifuge at 3000 rpm for 6 min. Remove the organic layer and add it to 100 |xL 0.2% ascorbic acid in MeOH, evaporate to dryness under vacuum while vortexing, reconstitute the residue in 200 |xL mobile phase, mix, filter, keep in ice, inject a 20 |xL aliquot. (Buffer was 27.6 g NaH2PO4 + 25.2 g sodium sulfite in 100 mL water, pH 6.1.) HPLCVARIABLES Column: 100 X 2 5 |xm Lichrosorb RP8 Mobile phase: MeCN: 100 mM citric acid 24:76 Flow rate: 0.5 Injection volume: 20 Detector: UV 350 CHROMATOGRAM Retention time: 9 Internal standard: demeclocycline (4) Limit of detection: 50 ng/mL OTHER SUBSTANCES Extracted: chlortetracycline, methacycline, oxytetracycline, tetracycline KEYWORDS serum REFERENCE
De Leenheer, A.R; Nelis, H.J.C.F. Doxycycline determination in human serum and urine by high-performance liquid chromatography. J.Pharm.ScL, 1979, 68, 999-1002

SAMPLE Matrix: bulk, formulations Sample preparation: Bulk. Prepare a 10-100 |xg/mL solution in buffer, inject an aliquot. Capsules, tablets. Prepare a 1 mg/mL solution of capsule contents or crushed tablets in

buffer, sonicate for 10 min, filter (0.45 ixm), dilute with buffer, inject an aliquot. (Buffer was 20 mM sodium perchlorate adjusted to pH 2.0 with perchloric acid.) HPLCVARIABLES Column: 250 X 4.6 5 jxm 100 A PLRP-S polystyrene-divinylbenzene (Polymer Laboratories) Mobile phase: MeCN: buffer 25:75 (Buffer was 20 mM sodium perchlorate adjusted to pH 2.0 with perchloric acid.) Flow rate: 1 Detector: UV 280 CHROMATOGRAM Retention time: 40 OTHER SUBSTANCES Simultaneous: impurities KEYWORDS capsules; tablets REFERENCE
Bryan, P.D.; Stewart, J.T. Chromatographic analysis of selected tetracyclines from dosage forms and bulk drug substance using polymeric columns with acidic mobile phases. J.Pharm.Biomed.Anal., 1994, 12, 675-692

SAMPLE Matrix: cell suspensions Sample preparation: 300 |xL Cell suspension + 300 |xL MeCN, vortex, centrifuge, inject a 10 jxL aliquot. HPLCVARIABLES Column: 125 X 4 Nucleosil 100 5CN Mobile phase: MeCN: THF: phosphate/citrate buffer 10:10:80 Injection volume: 10 Detector: UV 350 CHROMATOGRAM Retention time: 2.1 REFERENCE
Kersten, A.; Poitschek, C; Rauch, S.; Aberer, E. Effects of penicillin, ceftriaxone, and doxycycline on morphology of Borrelia burgdorferi. Antimicrob.Agents Chemother., 1995, 39, 1127-1133

SAMPLE Matrix: food Sample preparation: Condition a 100 mg Baker 10 C18 SPE cartridge by washing with MeOH, water, and 10 mL saturated aqueous Na2EDTA. Dissolve 5 g honey in 20 mL 100 mM pH 4.0 Na2EDTA-McIlvaine buffer, filter, apply to the SPE cartridge, wash with 20 mL water, air dry under vacuum for 5 min. Condition a Baker 10 COOH cartridge with ethyl acetate. Elute contents of C18 cartridge onto COOH cartridge with 50 mL ethyl acetate. Wash COOH cartridge with 10 mL MeOH, elute with 10 mL mobile phase, inject 100 |JLL aliquot. HPLCVARIABLES Column: 250 X 4.6 5 fxm Bakerbond C8 Mobile phase: MeOH: MeCN .10 mM aqueous oxalic acid 1:1.5:3

Flow rate: 1 Injection volume: 100 Detector: UV 350

CHROMATOGRAM

Retention time: 6 Limit of detection: 0.05 ppm

OTHER SUBSTANCES

Extracted: chlortetracycline, oxytetracycline, tetracycline

KEYWORDS

honey; SPE
REFERENCE
Oka, H.; Ikai, Y; Kawamura, N.; Uno, K.; Yamada, M.; Harada, K.; Uchiyama, M.; Asukabe, H., Mori, Y; Suzuki, M. Improvement of chemical analysis of antibiotics. IX. A simple method for residual tetracyclines analysis in honey using a tandem cartridge clean-up system. J.Chromatogr., 1987, 389, 417-426

SAMPLE

Matrix: food Sample preparation: Condition a 500 mg Baker-10 C18 SPE cartridge with 10 mL MeOH, 10 mL water, and 10 mL saturated aqueous disodium EDTA. Condition a 500 mg Baker10 COOH cartridge with MeOH: ethyl acetate 10:90. Dissolve 25 g honey in 50 mL 100 mM pH 4.0 disodium EDTA-Mcllvaine buffer, filter. Add the filtrate to the C18 SPE cartridge, wash with 20 mL water, wash with 400 \xL ethyl acetate, air dry under vacuum for 5 min, elute with 50 mL MeOH: ethyl acetate 10:90. Add a 5 mL aliquot to the COOH SPE cartridge, wash with 5 mL MeOH (?), elute with 10 mL mobile phase, inject a 100 |JLL aliquot.
HPLCVARIABLES

Column: 75 X 4.6 3 |xm Chemcosorb 3C8 (Chemco) Mobile phase: MeCN:MeOH: 10 mM aqueous oxalic acid 3:2:16, pH 3.0 Flow rate: 1 Injection volume: 100 Detector: UV 350
CHROMATOGRAM

Retention time: 9 Limit of detection: 0.1 ppm

OTHER SUBSTANCES

Extracted: chlortetracycline, demeclocycline (demethylchlortetracycline), methacycline, minocycline, oxytetracycline, tetracycline

KEYWORDS

honey; SPE
REFERENCE
Oka, H.; Ikai, Y.; Kawamura, N.; Uno, K.; Yamada, M.; Harada, K.; Suzuki, M. Improvement of chemical analysis of antibiotics. XIL Simultaneous analysis of seven tetracyclines in honey. J.Chromatogr., 1987, 400, 253-261

SAMPLE

Matrix: formulations

HPLCVARIABLES Column: 250 X 4.6 5 |xm Bakerbond phenylethyl Mobile phase: MeOHrIOO mM NaH2PO4 70:30 Flow rate: 0.8 Detector: UV 280 CHROMATOGRAM Retention time: 5.25 (doxycline hyelate) KEYWORDS injections; saline; water; stability-indicating REFERENCE
Stiles, M.L.; Allen, L.V., Jr.; Prince, S. J. Stability of various antibiotics kept in an insulated poucb during administration via portable infusion pump. Am.J.Health-Syst.Pharm., 1995, 52, 70-74

SAMPLE Matrix: formulations Sample preparation: Dissolve ointment in petroleum ether, add an equal volume of EtOH-.water 70:30, dilute with MeOH to 100 |xg/mL, inject a 10 |JLL aliquot. HPLCVARIABLES Column: 300 X 3.9 10 jxm LiChrosorb Si-60 Mobile phase: MeOH: water 5:95 containing 1.3 mM disodium citrate, 1 mM tetrabutylammonium bromide, 1.1 mM citric acid, and 8 mM EDTA. Flow rate: 1 Injection volume: 10 Detector: UV 254 CHROMATOGRAM Retention time: k' 0.56 OTHER SUBSTANCES Simultaneous: anhydrotetracycline, chlortetracycline, demeclocycline, epianhydrotetracycline, oxytetracycline, quatrimycin, rolitetracycline, tetracycline KEYWORDS ointment REFERENCE
Lingeman, H.; van Minister, H.A.; Beynen, J.H.; Underberg, W.J.; Hulshoff, A. High-performance liquid chromatographic analysis of basic compounds on non-modified silica gel and aluminium oxide with aqueous solvent mixtures. J.Chromatogr., 1986, 352, 261-274

SAMPLE Matrix: milk Sample preparation: Prepare a column as follows. Swirl Chelating Sepharose Fast Flow resin (Pharmacia) in its bottle, add it to a polypropylene column to give a bed volume of 1.0-1.2 mL, wash 3 times with 2 mL portions of water, wash with 2 mL 10 mM copper sulfate, wash with two 2 mL portions of water. Centrifuge 5 mL milk at 10 at 1500 g for 15 min, remove the lower layer and add it to 10 mL succinate buffer, mix, centrifuge at 1500 g for 30 min, add the supernatant to the column. Wash with 2 mL succinate buffer, wash with 2 mL water, wash with 2 mL MeOH, wash with 2 mL water, wash with 700 IxL citrate/phosphate buffer (be careful not to disturb bed), elute with 2.5 mL citrate/phosphate buffer (column is white and eluate is blue). Filter (Amicon Centricon 30,

MW 30000 cut-off; pre-washed by centrifuging with 2 mL water) while centrifiiging at 5000 g for 30-90 min, inject a 600 |JLL aliquot of the ultrafiltrate. (Prepare succinate buffer by dissolving 11.8 g succinic acid in 980 mL water, adjust pH to 4.0 with 10 M NaOH, make up to 1 L. Prepare the citrate/phosphate buffer by dissolving 12.9 g citric acid monohydrate, 10.9 g Na2HPO4, 37.2 g disodium EDTA dihydrate, and 29.2 g NaCl in 1 L water.) HPLCVARIABLES Column: 150 X 4.6 5 |xm PLRP-S (Polymer Labs) Mobile phase: Gradient. MeCN:MeOH: 10 mM oxalic acid 0:0:100 for 1 min, to 22:8:70 over 5 min, maintain at 22:8:70 for 11 min, return to initial conditions. Flow rate: 1 Injection volume: 600 Detector: UV 355 CHROMATOGRAM Retention time: 16.6 Limit of detection: 1.15 ng/mL Limit of quantitation: 2.22 ng/mL OTHER SUBSTANCES Extracted: chlortetracycline, demeclocycline, methacycline, minocycline, oxytetracycline, tetracycline Noninterfering: chloramphenicol, gentian violet, hydromycin B, ivermectin, spectinomycin, sulfa drugs KEYWORDS cow; SPE; ultrafiltrate REFERENCE
Carson, M.C. Simultaneous determination of multiple tetracycline residues in milk using metal chelate affinity chromatography J.AOAC Int., 1993, 76, 329-334

SAMPLE Matrix: tissue Sample preparation: Prepare an affinity column by filling a 10 mL column with 5 mL chelating Sepharose, allow to settle, wash with 20 mL 0.5% copper(II) sulfate solution, eliminate air bubbles by agitation, wash with 15 mL 50 mM pH 4 succinate buffer, do not allow to dry. Condition an Analytichem Bond Elut C18 SPE cartridge with 10 mL MeOH and 10 mL water, do not allow to dry. Homogenize 4 g minced kidney with 40 mL 50 mM pH 4 succinate buffer, sonicate for 10 min, centrifuge at 9000 rpm for 10 min, filter the supernatant through paper, repeat the extraction. Combine the supernatants and pass them through the affinity column at 5-7 mL/min, wash with 10 mL water, wash with 30 mL MeOH, wash with 20 mL water, elute with 50 mL 50 mM pH 4 succinate buffer containing 3.7% Titriplex III (ethylenedinitrilotetracacetic acid, disodium salt dihydrate). Add the eluate to the SPE cartridge at 5-7 mL/min, wash with 10 mL water, dry with air aspiration for 10 min, elute with 5 mL MeOHrMeCN 1:1, evaporate the eluate at 40 under a stream of nitrogen, dissolve the residue in 500 JJIL mobile phase, inject an aliquot. Protect from light through process. (The affinity columns may be re-used up to 15 times by washing with 20 mL water then 20 mL EtOH: water 20:80 then conditioning as described above.) HPLCVARIABLES Guard column: Perisorb RP-8 Column: two 300 X 100 5 |xm Chromspher C8 columns (cat. no. 28262) in series Mobile phase: MeCN: 10 mM pH 2 oxalic acid 20:80 Flow rate: 0.8 Detector: UV 365

CHROMATOGRAM Retention time: 26 Limit of quantitation: 30 ng/'g OTHER SUBSTANCES Simultaneous: ehlortetracycline, demethylchlortetracycline, methacycline, oxytetraeycline, tetracycline KEYWORDS kidney; SPE REFERENCE
Degroodt, J.M.; Wyhowski de Bukanski, B.; Srebrnik, S. Multiresidue analysis of tetracyclines in kidney by HPLC and photodiode array detection. J.Liq.Chromatogr., 1993, 16, 3515-3529

SAMPLE Matrix: tissue Sample preparation: Mince 0.1-0.3 g tissue with a scalpel and incubate at 37 with 0.5 mL water for 1 h. Add MeCN: 85% phosphoric acid: water 20:2:78 (muscle, renal medulla, lung) or MeOH: MeCN: 85% phosphoric acid .water 30:10:2:58 (renal cortex, liver) to a total volume of 1 mL, sonicate 30 min, filter (10 000 or 30 000 Da cutoff) by centrifuging at 2200 g for 30 min, inject 10-30 |xL aliquot of filtrate. HPLCVARIABLES Column: 220 X 2.1 Brownlee phenyl Spheri-5 MPLC cartridge Mobile phase: MeOH:MeCN:triethylamine:phosphoric acid:80 mM pH 2.4 sodium phosphate buffer 22.5:2.5:0.5:1.7:72.8 Column temperature: 60 Flow rate: 0.3-0.4 Injection volume: 10-30 Detector: UV 268; UV 345 CHROMATOGRAM Retention time: 6 Limit of detection: <5-10 ng/g KEYWORDS cow; muscle; renal cortex; renal medulla; liver; lung REFERENCE
Riond, J.L.; Hedeen, K.M.; Tyezkowska, K.; Riviere, J.E. Determination of doxycycline in bovine tissues and body fluids by high-performance liquid chromatography using photodiode array ultraviolet-visible detection. J.Pharm.ScL, 1989, 78, 44-47

ANNOTATED BIBLIOGRAPHY

Prevosto, J.M.; Beraud, B.; Cheminel, V.; Gaillard, Y; Mounier, C; Chaulet, J.F. Determination of doxycycline in human plasma and urine samples by high performance liquid chromatography. Application for drug monitoring in malaria chemoprophylaxis. Ann.Biol.Clin.(Paris), 1995, 53, 29-32 Colmenero, J.D.; Fernandez-Gallardo, L.C; Agiindez, J.A.G.; Sedeno, J.; Benitez, J.; Valverde, E. Possible implications of doxycycline-rifampin interaction for treatment of brucellosis. Antimicrob.Agents Chemother., 1994, 38, 2798-2802 [extracted rifampin; plasma; serum; papaverine (IS); LOQ 200 ng/mL] Hoogmartens, J.; Khan, N.H.; Vanderhaeghe, H.; Van der Leeden, A..L.; Oosterbaan, M.; Veld-Tulp, G.L.; Plugge, W.; Van der Vlies, C; Mialanne, D.; et al. A collaborative study of the analysis of doxycycline

hyclate by high-performance liquid chromatography on polystyrene-divinylbenzene packing materials. J.Pharm.Biomed.Anal, 1989, 7, 601-610 Nieder, M.; Jaeger, H. Selective quantification of doxycycline in human plasma and urine with optimized chromatography. Chromatographia, 1988, 25, 526-530 [column temp 30; plasma; urine; SPE; demeclocycline (IS); pharmacokinetics; non-interfering other tetracyclines, caffeine, nicotine, salicylic acid; LOQ 125 ng/mL] Dihuidi, K.; Kucharski, M.J.; Roets, E.; Hoogmartens, J.; Vanderhaeghe, H. Quantitative analysis of doxycycline and related substances by high-performance liquid chromatography. J.Chromatogr., 1985, 325, 413424 [column temp 60; bulk; tablets; capsules; simultaneous impurities, methacycline, oxytetracycline] Bocker, R. Rapid analysis of doxycycline from biological samples by high-performance liquid chromatography. J.Chromatogr., 1980, 187, 439-441 [whole blood; serum; tissue; mouse; liver] De Leenheer, A.P.; Nelis, H.J.C.F. Reversed-phase high-performance liquid chromatography of doxycycline. J.Chromatogr., 1977, 140, 293-299